Time between antiretroviral treatment interruption (ART-i) and the detection of viremia in plasma is a direct reflection of the size of the functional reservoir. Despite controversies about residual viremia, the persistence of active but limited HIV-1 and SIV replication during antiretroviral therapy (ART), data from reports where patients experienced viral rebound after months without ART corroborate the hypothesis that the latent reservoirs are composed of only latent or non-replicating forms of virus. Therefore, reducing the size of the replication competent functional reservoirs would delay or prevent viral rebound. In this project we will characterize and quantitate the latent SIV reservoir in blood and tissues during ART suppression and during successive steps after stopping ART (before detectable plasma viral load (pVL), ascending pVL, and set point post rebound). Quantitation of cells harboring replication competent SIV genomes will define the cellular reservoirs and identify the anatomic locations where these reservoirs are located. Performing these experiments during the early stage of ART-i will determine the contribution of each reservoir to viral rebound. These studies will also identify the distinct reactivating properties of resting memory CD4+ T cells (rmCD4s) and the three different macrophages (M) subsets: monocytes (which are not a M per se, but the blood precursors of a portion of tissue M); monocyte-derived M in tissues; and resident, or fetal tissue-derived M. In addition to using immunohistochemistry (IHC) and in situ hybridization (ISH) to characterize the virus-producing cells in tissue sections during viral rebound, we will analyze the functional reservoir ex vivo comparing five different techniques (discussed below). We hypothesize that: 1) Both rmCD4s and tissue M are latently infected in vivo and form the functional viral reservoir that can be reactivated when ART is interrupted. 2) Rebound viruses can originate from multiple cell types and tissues. 3) Results from the assays here proposed will predict the kinetics of viral rebound post ART-i. 4) In vivo CD4+ T cell depletion will decrease the latent CD4+ reservoir and delay rebound after ART-i.